Radioactive metal-labeled anti-cadherin antibody

ABSTRACT

A radioactive metal-labeled anti-cadherin antibody which is obtained by binding a radioactive metallic element to an anti-cadherin antibody via a metal-chelating reagent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/578,462filed Aug. 10, 2012, allowed and incorporated herein by reference, whichis a National Stage of PCT/JP11/052,759 filed Feb. 9, 2011 and claimsthe benefit of JP 2010-028028 filed Feb. 10, 2010.

TECHNICAL FIELD

The present invention relates to a radioactive metal-labeledanti-cadherin antibody which highly specifically accumulates in cancercells, and to a cancer therapeutic agent and a cancer diagnostic agenteach containing the antibody.

BACKGROUND ART

There is keen demand for new cancer therapy for the treatment of cancer,which is now the leading cause of death. Currently, cancer therapiessuch as surgical therapy, radiotherapy, and chemotherapy (by use of ananti-cancer agent) are employed. Even after surgery, an anti-canceragent is employed in postoperative therapy.

Currently employed anti-cancer agents include an alkylating agent, anantimetabolite, an alkaloide anti-cancer agent, an antibioticanti-cancer agent, and a platinum agent. The treatment effects of theseagents are not completely satisfactory. Some agents are not cancercell-specific and frequently cause adverse side effects, which isproblematic. Under such circumstances, there is demand for developmentof more effective anti-cancer agents.

Meanwhile, cadherin is a Ca²⁺-dependent adhesion molecule which isexpressed on the cell surface. Examples of known cadherin speciesinclude classic cadherins such as E cadherin, N cadherin, and P cadherin(CDH3); as well as protocadherin, and desmosomal cadherin. Thesecadherins are known to bind homophylicly, to form an adherence junction,and to link to the cytoskeletal system (actin filaments) viaintracellular catenin and are considered to control cell adhesion bysuch a mechanism.

In addition to cell adhesion, cadherin is thought to relate toembryogenesis, morphogenesis, synaptogenesis, synaptic plasticity, andinfiltration and metastasis of cancer. Thus, an anti-cadherin antibodyis reported to be useful for cancer therapy (Patent Documents 1 to 3).

PRIOR ART DOCUMENTS Patent Documents Patent Document 1: Japanese Kohyo(PCT) Patent Publication No. 2005-522982 Patent Document 2: JapaneseKohyo (PCT) Patent Publication No. 2008-538909 Patent Document 3:Japanese Kohyo (PCT) Patent Publication No. 2009-528257 SUMMARY OF THEINVENTION Problems to be Solved by the Invention

However, the anti-cancer effect of the anti-cadherin antibody is notsatisfactory, and there has been demand for development of a more potentcancer therapeutic agent.

Thus, an object of the present invention is to provide a radioactivemetal-labeled anti-cadherin antibody which can be highly accumulated incancer tissue. Another object is to provide a cancer therapeutic agentwhich contains the antibody as an active ingredient and which exhibitshigh anti-cancer effect. Still another object is to provide a cancerdiagnostic agent which can predict the efficacy of a cancer therapeuticagent and confirm the therapeutic effect thereof.

Means for Solving the Problems

The present inventors have conducted extensive studies to attain theaforementioned objects, and have found that a radioactive metal-labeledanti-cadherin antibody in which a radioactive metallic element is boundto a anti-cadherin antibody via a metal-chelating reagent is accumulatedspecifically in the cancer tissue of a cancer-bearing animal, and thatthe anti-cancer effect thereof is particularly remarkably enhanced ascompared to the unlabeled anti-cadherin antibody-administration group.The present invention has been accomplished on the basis of thesefindings.

Accordingly, the present invention provides a radioactive metal-labeledanti-cadherin antibody which is obtained by binding a radioactivemetallic element to an anti-cadherin antibody via a metal-chelatingreagent, and a cancer therapeutic agent and a cancer diagnostic agenteach containing, as an active ingredient, the radioactive metal-labeledanti-cadherin antibody.

The present invention also provides the radioactive metal-labeledanti-cadherin antibody for use in the treatment or diagnosis of cancer.

The present invention also provides use of the radioactive metal-labeledanti-cadherin antibody for producing a cancer therapeutic agent or acancer diagnostic agent.

The present invention also provides a method for the treatment ordiagnosis of cancer, containing administering an effective amount of theradioactive metal-labeled anti-cadherin antibody to a subject in needthereof.

Effects of the Invention

The cancer therapeutic agent containing, as an active ingredient, theradioactive metal-labeled anti-cadherin antibody of the presentinvention is highly accumulated in cancer tissue and exhibits highcancer tissue-shrinking effect. Therefore, by use of the cancertherapeutic agent, cancer therapy can be effectively performed withoutcausing adverse side effects. Also, by use of the cancer diagnosticagent of the present invention, the efficacy of the cancer therapeuticagent of the present invention can be predicted, and the therapeuticeffect thereof can be confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Graphs showing the result of the affinity of antibodies evaluatedby flow cytometry.

FIG. 2 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2016 antibody (adding ratio of1:10) 96 hours after administration thereof.

FIG. 3 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2016 antibody (adding ratio of1:3) 96 hours after administration thereof.

FIG. 4 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2029 antibody (adding ratio of1:10) 96 hours after administration thereof.

FIG. 5 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2029 antibody (adding ratio of1:3) 96 hours after administration thereof.

FIG. 6 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2025 antibody (adding ratio of1:10) 96 hours after administration thereof.

FIG. 7 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2025 antibody (adding ratio of1:3) 96 hours after administration thereof.

FIG. 8 Bio-distribution of ⁶⁷Ga-DOTA-PPMX2025 antibody in case of theadded antibody-DOTA ratios of 1:1, 1:3, and 1:10 96 hours afteradministration thereof.

FIG. 9 Bio-distribution of ¹¹¹In-DOTA-PPAT-052-27c antibody (addingratio of 1:3) 96 hours after administration thereof.

FIG. 10 Bio-distribution of ¹¹¹In-DOTA-PPAT-052-27c antibody (addingratio of 1:3) 48 hours after administration thereof.

FIG. 11 Bio-distribution of ¹¹¹In-DOTA-PPAT-052-28c antibody (addingratio of 1:3) 48 hours and 96 hours after administration thereof.

FIG. 12 Anti-tumor effect of ⁹⁰Y-DOTA-PPMX2029 antibody (adding ratio of1:3) in xenograft model.

FIG. 13 Anti-tumor effect of ⁹⁰Y-DOTA-PPAT-052-27c antibody (addingratio of 1:3) in xenograft model.

FIG. 14 Photoimages showing the results of immunohistochemical stainingfor confirming CDH3 protein expression.

MODES FOR CARRYING OUT THE INVENTION

The radioactive metal-labeled anti-cadherin antibody of the presentinvention is a labeled anti-cadherin antibody to which a radioactivemetallic element is bound to via a metal-chelating reagent. The cancertherapeutic agent or the cancer diagnostic agent contains theradioactive metal-labeled anti-cadherin antibody.

The anti-cadherin antibody is not particularly limited, so long as theantibody specifically binds to cadherin. Examples of cadherin include Ecadherin, N cadherin, and P cadherin. Of these, P cadherin is morepreferred.

The anti-cadherin antibody encompasses a monoclonal antibody, apolyclonal antibody, an antibody maintaining ability of bindingspecifically to an antigenic determinant group and variants andderivatives of an antibody such as T-cell receptor fragment.

The type of the anti-cadherin antibody is not particularly limited, andthere may be appropriately employed antibodies such as a mouse antibody,a human antibody, a rat antibody, a rabbit antibody, a sheep antibody, acamel antibody, and a chicken antibody; and gene recombinant antibodieswhich are intentionally modified so as to reduce the hetero-antigenicityto human such as a chimeric antibody and a humanized antibody. Therecombinant antibody may be produced through a known method. Thechimeric antibody is an antibody formed of variable regions of a heavychain and a light chain of a mammalian antibody other than humanantibody, for example mouse antibody and constant regions of a heavychain and a light chain of a human antibody and may be produced bylinking a DNA fragment encoding the variable region of the mouseantibody to a DNA fragment encoding the constant region of the humanantibody, incorporating the resultant fragment into an expressionvector, and incorporating the vector into host cells (see, for example,Cabilly S. et al., Proc. Natl. Acad. Sci. USA, 1984, 81(11) 3273-7;Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81(21), 6851-5; andEuropean Patent Application Laid-Open No. 171496). The humanizedantibody, which is also called a reshaped antibody, is an antibodyproduced through transplantation of a complementarity determining region(CDR) of a mammalian antibody other than human antibody, e.g., a mouseantibody into a CDR of a human antibody, and gene recombinationtechniques therefor are generally known. Specifically, a DNA sequenceincluding a CDR of a mouse antibody linked to a framework region (FR) ofa human antibody is synthesized through PCR using severaloligonucleotides which are produced to have overlapped part at an endthereof. The thus-obtained DNA fragment is linked to a DNA fragmentencoding the constant region of the human antibody, subsequently theresultant fragment is incorporated into an expression vector and thevector is incorporated into host cells to thereby produce the humanizedantibody (see EP239400 A and WO 96/02576 A). The FR of the humanantibody linked via the CDR is selected from FRs having a CDR whichforms a suitable antigen-binding site. If needed, an amino acid in theFR of the variable region of the antibody may be substituted such that aCDR of the reshaped antibody forms an appropriate antigen-binding site(Sato, K. et al., Cancer Res., 1993, 53, 851-856).

The amino acid sequence of the chimeric antibody or humanized antibodypreferably has an identity of 1005 to that of the Vh or Vl region ofcDNA expressing a deposited hybridoma. Due to genetic modification, anantibody having an identity in amino acid sequence of 90% or higher isalso preferred. In the process of humanization or chimerization, therehas been conventionally carried out such controlled residue substitutionfor improving binding to an antigen. Such an antibody having a partiallymodified sequence is essentially considered to be an antibodyoriginating from the original hybridoma.

Methods for producing a chimeric antibody and a humanized antibody basedon a genetic engineering technique have been already known.Specifically, the Vh and VL sequences of a monoclonal antibody servingas a confirmed group is genetically modified, and then chimerization orhumanization is performed through a routine technique.

The method for recovering a human antibody is also known. In oneprocedure, human lymphocytes are sensitized in vitro with an antigen ofinterest or with cells expressing the antigen, and the thus-sensitizedlymphocytes are fused with human myeloma cells, e.g., U266, to therebyproduce a human antibody of interest having a binding activity to theantigen (see JP-B-1989-59878). Alternatively, a human antibody ofinterest may be recovered through immunization, with an antigen ofinterest, of a transgenic animal having a complete repertory of thehuman antibody gene (see WO 93/12227, WO 92/03918, WO 94/02602, WO94/25585, WO96/34096, and WO96/33735). Also known is a technique forrecovering a human antibody through panning by use of a human antibodylibrary. In one procedure, a variable region of a human antibody isexpressed as a single-chain antibody (scFv) on the phage surface throughthe phage display method, and a phage which binds the antigen can beselected. Through gene analysis of the thus-selected phage, a DNAsequence encoding the variable region of the human antibody which bindsto the antigen can be determined. When the DNA sequence of the scFvwhich binds the antigen is elucidated, an appropriate expression vectorcan be produced from the sequence, whereby a human antibody of interestcan be recovered. These methods are widely known (see WO 92/01047, WO92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO95/15388).

These anti-cadherin antibodies may be a low molecule antibody such as anantibody fragment, a modified antibody or the like, so long as theability of recognizing the entire or a part of the protein encoded bythe cadherin gene is maintained. Examples of the antibody fragmentinclude Fab, Fab′, F(ab′)2, Fv, and Diabody. Such an antibody fragmentmay be produced by constructing a gene encoding the antibody fragment,incorporating the gene into an expression vector, and expressing thevector in appropriate host cells (see, for example, Co, M. S. et al., J.Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., MethodsEnzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., MethodsEnzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121,652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; andBird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

As a modified antibody, an antibody which is bound to any of variousmolecules such as polyethylene glycol (PEG) may be used. Such a modifiedantibody may be produced through chemical modification of the obtainedantibody. The antibody modification technique has already beenestablished in the art.

In the present invention, there may be also employed a sugar chainmodified antibody for potentiating cytotoxic activity. Techniques ofmodifying the sugar chain in an antibody have already been known (e.g.,WO 00/61739 and WO 02/31140).

The anti-cadherin antibody of the present invention also encompasses amulti-specific antibody having specificity to two or more differentantigens. A typical example of such a molecule may be one which can bindtwo antigens (i.e., a bi-specific antibody). The “multi-specificantibody” of the present invention includes an antibody having aspecificity to two or more (e.g., three) antigens. The multi-specificantibody may be a full-length antibody or a fragment of such an antibody(e.g., F(ab′)₂ bi-specific antibody).

The anti-cadherin antibody of the present invention and the antibodyfragment thereof may be produced through any suitable method such as invivo, cultured cells, in vitro translation reaction, and recombinant DNAexpression system.

Techniques of producing monoclonal antibodies and antibody-producingcells (hybridomas) are generally known in the art (Campbell, “MonoclonalAntibody Technology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;and St. Groth et al., J. Immunol. Methods 35: 1-21, 1980). In onespecific procedure, a protein or a fragment thereof encoded by acadherin gene serving as an immunogen is subcutaneously orintraperitoneally injected for immunization to any animal (e.g., mouseor rabbit) which is known to produce an antibody. In immunization, anadjuvant may be employed, and such an adjuvant is well known in the art.

The polyclonal antibody may be produced by isolating an anti-serumcontaining antibodies from an immunized animal and screening for thepresence of an antibody having a target specificity through a techniquewell known in the art (e.g., ELISA, Western blotting, orradioimmunoassay).

The monoclonal antibody may be produced by removing spleen cells from animmunized animal and fusing the cells with myeloma cells, to therebyproduce hybridomas which can produce monoclonal antibodies. Hybridomacells producing an antibody which can recognize a protein of interest ora fragment thereof may be selected based on a technique well known inthe art (e.g., ELISA, Western blotting, or radioimmunoassay). Then, thehybridoma secreting an antibody of interest is cloned, and the obtainedcells are cultured under appropriate conditions. The thus-secretedantibody is recovered and purified through a method well known in theart (e.g., ion-exchange column chromatography or affinitychromatography). In an alternative procedure, a human monoclonalantibody may be produced by use of a xenomouse strain (see Green, J.Immunol. Methods 231: 11-23, 1999; and Wells, Eek, Chem. Biol. 2000August; 7(8): R185-6). Currently, monoclonal antibody production basedon phage display involving no immunization is carried out. Themonoclonal antibody of the present invention is asingle-molecular-species antibody produced by single-species ofantibody-producing cells or a DNA fragment obtained therefrom andencoding the antibody. The monoclonal antibody may be produced throughany of the aforementioned methods.

The DNA fragment encoding a monoclonal antibody can be readily isolatedand sequenced through a routine method (e.g., by use of anoligonucleotide probe which can binds specifically to genes encoding theheavy chain and light chain of the monoclonal antibody). A hybridomacell is a preferred starting material for producing such a DNA fragment.After isolation, such a DNA fragment is inserted into an expressionvector, and the vector is recombined to host cells such as E. colicells, monkey COS cells, Chinese hamster ovary (CHO) cells or myelomacells in which no immunoglobulin is produced unless the cells aretransformed. The monoclonal antibody of interest is produced by therecombinant host cells. In an alternative mode, an antibody or anantibody fragment can be isolated from an antibody phage libraryproduced through a technique of McCafferty et al. (Nature 348: 552-554(1990)).

The host cell employed for monoclonal antibody expression is preferablya mammal-origin host cell. A host cell most suited to a monoclonalantibody to be expressed may be selected. The host cell is not limitedand typical examples thereof include CHO-originating cell line (Chinesehamster ovary cell), CV1 (monkey kidney), COS (CV1 derivative expressingSV40T antigen), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma),293 (human kidney), and 293T (293 derivative expressing SV40T antigen).The host cell system may be obtained from a commercial facility, theAmerican Tissue Culture Collection (ATCC), or an organization whichpublished a relevant document.

The host cell is preferably a dhfr gene expression-defectiveCHO-originating cell line (deletion in dhfr gene expression) or SP2/0(see Urland, G. et al., Effect of gamma rays at the dihydrofolatereductase locus: deletions and inversions; Somat. Cell. Mol. Genet. Vol.12, 1986, p. 5555-566; and Schulman, M. et al., A better cell line formaking hybridomas secreting specific antibodies, Nature Vol. 276, 1978,p. 269-270). The host cell is more preferably a DHFR-deleted CHO.Transfection of a plasmid into host cells may be performed through anytechnique. Transfection technique is not limited and specific examplesthereof include transfection (including calcium phosphate method, DEAEmethod, lipofection, and electroporation), DNA incorporation by use ofan envelope (e.g., Sendai virus), micro-injection, and infection by useof a viral (e.g., retrovirus or adenovirus) vector (see CurrentProtocols in Molecular Biology, Chapter 9 Introduction of DNA intoMammalian Cells, John Wiley and Sons, Inc.). Among them, incorporationof a plasmid into host cells through electroporation is particularlypreferred.

The recognition site in cadherin of the anti-cadherin antibody of thepresent invention is preferably a region from 1 to 655 of SEQ ID NO: 2.

The anti-cadherin antibody of the present invention is preferablyproduced from a hybridoma PPMX2016, PPMX2025, PPMX2029, PPAT-052-02,PPAT-052-03, PPAT-052-09, PPAT-052-24, PPAT-052-25, PPAT-052-26, orPPAT-052-28, or an transgenic CHO cell line PPAT-052-27c, PPAT-052-02c,PPAT-052-03c, PPAT-052-09c, PPAT-052-21c, PPAT-052-24c, PPAT-052-25c,PPAT-052-26c, PPAT-052-28c, or PPAT-052-29c. In the presentspecification, the numbers attached to PPMX or PPAT are given to eithercorresponding antibody-producing cells or antibodies produced by theantibody-producing cells.

A radioactive metal which is bound to the anti-cadherin antibody ispreferably a cytotoxic radioactive metal when the radioactivemetal-labeled anti-cadherin antibody is used as a cancer therapeuticagent, and a non-cytotoxic radioactive metal when the radioactivemetal-labeled anti-cadherin antibody is used as a cancer diagnosticagent.

Examples of the cytotoxic radioactive metal include yttrium-90 (⁹⁰Y),rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re), copper-67 (⁶⁷Cu), iron-59(⁵⁹Fe), strontium-89 (⁸⁹Sr), gold-198 (¹⁹⁸Au),)mercury-203 (²⁰³Hg),lead-212 (212Pb),)dysprosium-165 (¹⁶⁵Dy), ruthenium-103 (¹⁰³Ru),bismuth-212 (212Bi), (²¹²Bi) bismuth-213 (²¹³Bi),)holmium-166 (¹⁶⁶Ho),samarium-153 (153Sm), and lutetium-177 (¹⁷⁷Lu).

Among these radioactive metals, ⁹⁰Y, ¹⁵³Sm, and ¹⁷⁷Lu are preferred,from the viewpoints of half-life, radiation energy, ease of labelingreaction, percent of labeling, complex stability, etc.

A non-cytotoxic radioactive metal suitably employed in a cancerdiagnostic agent is not limited and examples thereof includetechnetium-99m (^(99m)Tc), indium-111)indium-113m (^(1l3m)In),gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga), thallium-201 (²⁰¹Tl), chromium-51(⁵¹Cr), cobalt-57 (⁵⁷Co), cobalt-58 (⁵⁸Co), cobalt-60 (⁶⁰Co),strontium-85 (⁸⁵Sr), mercury-197 (¹⁹⁷Hg), and copper-64 (⁶⁴Cu).

For bonding a radioactive metallic element to the anti-cadherinantibody, in a preferred mode, a metal-chelating reagent is reacted withthe anti-cadherin antibody, and the product is further reacted with aradioactive metallic element, to thereby form a complex. In thethus-produced modified antibody, the radioactive metallic element isbound to the anti-cadherin antibody via the metal-chelating reagent.

Examples of the metal-chelating reagent for forming such a complexinclude (1) quinoline derivatives such as 8-hydroxyquinoline,8-acetoxyquinoline, 8-hydroxyquinaldine, oxyquinoline sulfate,O-acetyloxine, O-benzoyloxine, O-p-nitrobenzoyloxine, and quinolonecompounds having a quinoline skeleton (e.g., norfloxacin, ofloxacin,enoxacin, ciprofloxacin, lomefloxacin, tosfloxacin, fleroxacin, andsparfloxacin); (2) compounds such as chloranilic acid, aluminon,thiourea, pyrogallol, cupferron, Bismuthiol (II), galloyl gallic acid,thiolide, 2-mercaptobenzothiazole, and tetraphenylarsonium chloride; (3)ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), and compounds having a similar skeleton(dihydroxyethylglycine, diaminopropanolte traacetic acid,ethylenediamine diacetic acid, ethylenediaminedipropionic acidhydrochloride, hydroxyethylethylenediaminetriacetic acid,ethylenediaminetetrakis(methylenesulfonic acid), glycol etherdiaminetetraacetic acid, hexamethylenediaminetetraacetic acid,hydroxyethyliminodiacetic acid, iminodiacetic acid,diaminopropanetetraacetic acid, nitrilotriacetic acid,nitrilotripropionic acid, nitrilotris(methylenesulfonic acid) trisodiumsalt, triethylenetetraminehexaacetic acid, methyl DTPA, cyclohexyl DTPA,aminobenzyl EDTA, isothiocyanobenzyl EDTA, isothiocyanobenzyl DTPA,methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA,maleimidopropylamidobenzyl EDTA, maleimidopentylamidobenzyl EDTA,maleimidodecylamidobenzyl EDTA, maleimidopentylamidobenzyl DTPA, andmaleimidodecylamidobenzyl DTPA); and(4)1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA),1,4,7,10-tetraazacyclododecane (Cyclen),1,4,8,11-tetraazacyclotetradecan (Cyclam), isothiocyanobenzyl DOTA, andisothiocyanobenzyl NOTA.

Among these metal-chelating reagents, isothiocyanobenzyl DOTA,methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA arepreferred, from the viewpoints of ease of incorporation reaction ofmetal-chelate to antibody, percent of labeling, complex stability, etc.

The radioactive metallic element may be bound to the anti-cadherinantibody through a routine method. In one procedure, a metal-chelatingreagent is reacted with an anti-cadherin antibody, to thereby prepare alabel precursor, and the precursor is reacted with a radioactivemetallic element.

In the cancer therapeutic agent and cancer diagnostic agent of thepresent invention, the ratio by mole of anti-cadherin antibody tometal-chelating reagent is important for accumulation in cancer cellsand anti-cancer effect. The mole ratio (anti-cadherin antibody:chelatingreagent) is preferably 1:0.1 to 1:4.5, more preferably 1:0.5 to 1:3. Inorder to attain such mole ratios, the anti-cadherin antibody and thechelating reagent are preferably added to react at a ratio of 1:0.1 to1:less than 5, particularly preferably 1:1 to 1:3. The number of chelatemolecule(s) per anti-cadherin antibody may be calculated by measuringmolecular weight through MALDI-TOF mass analysis or a similar technique,and comparing the molecular weight of an un-modified antibody to that ofa modified antibody (U.S. Pat. No. 7,514,078, Lu et al., J. Pharm. Sci.94(4), 2005, p. 788-797, and Tedesco et al., J. Clin. Onco. 23 (16S),2005, 4765). Alternatively, the number of chelate molecule(s) peranti-cadherin antibody may be determined through chelatometrictitration. One known method employs an alkaline earth metal colorimetricreagent (arsenazo III) (Bradyr et al., Nucl. Med. Biol. 31, 795-802,2004, and Dadachova et al., Nucl. Med. Biol. 26, 977-982, 1999).

The cancer therapeutic agent or cancer diagnostic agent of the presentinvention may be provided as a labeled formulation or a kit formulationcontaining a label precursor. Either formulation may be employed in thepresent invention. In the case of labeled formulation, a cancertherapeutic agent or a cancer diagnostic agent containing a labeledanti-cadherin antibody may be administered as is. In the case of a kitformulation, the agent may be administered after labeling with aradioactive metallic element of interest.

The anti-cadherin antibody containing a radioactive metallic elementbound thereto highly accumulates in cancer tissue and exhibits highcancer cell-toxic activity. Thus, the antibody is a useful cancertherapeutic agent which less damages the tissue other than cancer tissueand which has high safety. Also, the anti-cadherin antibody containing aradioactive metallic element bound thereto has an anti-cancer activityremarkably higher than that of a corresponding anti-cadherin antibody.The anti-cancer activity is remarkably high particularly when the moleratio of antibody to chelating agent is 1:0.1 to 1:4.5.

The cancer therapeutic agent of the present invention may be used incombination with another anti-cancer agent. Examples of such ananti-cancer agent include an alkylating agent, an antimetabolite, amicrotubule inhibitor, an antibiotic anti-cancer agent, a topoisomeraseinhibitor, a platinum agent, a molecular target drug, a hormone agent,and a biologics. Examples of the alkylating agent include nitrogenmustard anti-cancer agents (e.g., cyclophosphamide), nitrosoureaanti-cancer agents (e.g., ranimustine), and dacarbazine. Examples of theantimetabolite include 5-FU, UFT, carmofur, capecitabine, tegafur, TS-1,gemcitabine, and cytarabine. Examples of the microtubule inhibitorinclude alkaloid anti-cancer agents (e.g., vincristine) and taxaneanti-cancer agents (e.g., docetaxel and paclitaxel). Examples of theantibiotic anti-cancer agent include mitomycin C, doxorubicin,epirubicin, daunorubicin, and bleomycine. Examples of the topoisomeraseinhibitor include irinotecan and nogitecan having topoisomerase Iinhibiting activity and etoposide having topoisomerase II inhibitingactivity. Examples of the platinum agent include cisplatin, paraplatin,nedaplatin, and oxaliplatin. Examples of the molecular target druginclude trastuzumab, rituximab, imatinib, gefitinib, erlotinib,bevacizumab, bortezomib, sunitinib, and sorafenib. Examples of thehormone agent include dexamethasone, finasteride, and tamoxifen.Examples of the biologics include interferons α, β, and γ andinterleukin 2.

The cancer therapeutic agent of the present invention may be used incombination with a cancer therapy. Examples of the cancer therapyinclude surgery, radiation therapies (including gamma knife therapy,Cyber knife therapy, boron neutron capture therapy, and protonbeam/heavy ion beam therapy), MR-guided focused ultrasound surgery,cryotherapy, radiofrequency ablation, percutaneous ethanol injectiontherapy, and embolotherapy.

The cancer therapeutic agent of the present invention is effective onvarious cancers of a mammal (including a human). Examples of the targetcancer include carcinomas such as pharyngeal cancer, laryngeal cancer,tongue cancer, lung cancer, breast cancer, esophageal cancer, stomachcancer, colorectal cancer, uterine cancer, ovarian cancer, liver cancer,pancreatic cancer, gallbladder cancer, kidney cancer, prostatic cancer,malignant melanoma, and thyroid cancer; and sarcomas such asosteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcome,liposarcoma, angiosarcoma, fibrosarcoma, leukemia, malignant lymphoma,and myeloma.

The cancer therapeutic agent of the present invention may be dissolvedin an aqueous solution, preferably a physiologically adaptable buffersuch as Hanks' solution, Ringer's solution, or buffered physiologicalsaline. Also, the therapeutic agent may have the form of suspension,solution, emulsion, or the like in an oily or aqueous vehicle.

The dosage of the cancer therapeutic agent of the present invention,which varies in accordance with the symptom, administration route, bodyweight, age, etc. of a patient in need thereof, is preferably, forexample, 37 to 3,700 MBq for one treatment of adult.

The cancer therapeutic agent of the present invention is generallyadministered parenterally. For example, the cancer therapeutic agent isinjected (e.g., subcutaneously, intravenous, intramuscle,intraperitoneally) or administered transdermally, transmucosally,transnasally, transplumonarily, etc.

The cancer diagnostic agent of the present invention may be used intumor imaging. In the case where a patient has a tumor in which CDH3protein is expressed, the cancer diagnostic agent of the presentinvention accumulates in the tumor. Thus, the tumor can be imaged bydetecting radiation by means of an apparatus such as a single photonemission computed tomograph (SPECT), a positron emission tomograph(PET), or a scintillation camera. For example, by use of the cancerdiagnostic agent of the present invention, the therapeutic effect of thecancer therapeutic agent of the present invention can be predictedbefore administration of the therapeutic agent. The diagnostic agent isadministered to a patient before the treatment, and the tumor is imaged.When high accumulation is observed, the superior effect of thetherapeutic agent can be predicted as potent. The diagnostic agent maybe used for determining the therapeutic effect. The diagnostic agent ofthe present invention is administered to a patient who has received thetreatment with the therapeutic agent of the present invention or anyother treatment so as to image the tumor. Through monitoring thetime-dependent variation in accumulation of the diagnostic agent, theexpansion or shrinkage of the tumor over time can be observed.

The antibody for use as a diagnostic agent preferably recognizes anepitope competitive to a therapeutic agent. More preferably, theantibody recognizes the same epitope as that recognized by thetherapeutic agent. Most preferably, the therapeutic agent and thediagnostic agent are the same antibody.

The cancer diagnostic agent of the present invention is generallyadministered to a subject intravenously. However, the cancer diagnosticagent may also be administered arterially. The dosage thereof, whichvaries in accordance with the symptom, administration route, bodyweight, age, etc. of a patient in need thereof, is preferably, forexample, 37 to 1,120 MBq for one treatment of adult.

EXAMPLES

The present invention will next be described in detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1 Production of Soluble CDH3 Antigen

Soluble CDH3 (sCDH3) protein in which the C-terminal transmembraneregion had been deleted was produced to serve as an immunogen forproducing an anti-CDH3 antibody.

(1) Production of Soluble CDH3 Antigen Expression Vector

PCR was performed by use of a CDH3 full-length cDNA as a template and aforward primer (SEQ ID NO: 3: CGCGGTACCATGGGGCTCCCTCGT, (hCDH3FullFW))and a reverse primer (SEQ ID NO: 4: CCGTCTAGATAACCTCCCTTCCAGGGTCC,(hCDH3SolbRV)), which had been designed so as to amplify a segmentcorresponding to the CDH3 extracellular region (1-654 in SEQ ID NO: 2,hereinafter referred to as sCDH3cDNA). The reaction was performed by useof KOD-Plus (product of Toyobo) and under the following conditions: 94°C.-15 sec, 55° C.-30 sec, and 68° C.-90 sec (30 cycles)).

After completion of the PCR reaction, the reaction mixture was subjectedto agarose gel electrophoresis, and a gel piece containing a band of atarget size (about 2.0 kbp) was cut out. The target sCDH3cDNA wasrecovered from the gel piece by use of a QIA quick gel extraction kit(product of Quiagen).

In order to insert sCDH3cDNA into an expression vector pEF4/myc-HisB,sCDH3cDNA was treated with two restriction enzymes KpnI and XbaI. Thethus-obtained fragment was inserted into pEF4/myc-HIsB which had beentreated with the same restriction enzymes KpnI and XbaI, by use of T4DNA ligase through a routine technique, whereby an expression vectorpEF4-sCDH3-myc-His was yielded.

(2) Expression of Soluble CDH3 Protein

According to a protocol of an FuGENE6 transfection reagent, 8×10⁵ CHOcells were inoculated to a 10-cm-diameter dish on the day beforetransfection, and the cells were cultured overnight. Thereafter, anexpression vector pEF4-sCDH3-myc-His (8 μg) and an FuGENE6 regent (16were mixed with serum-free RPMI 1640 medium (400 μL), and the mixturewas allowed to stand at room temperature for 15 minutes. The resultantmixture was added to the cell culture liquid for transfection. Two daysafter transfection, cloning was performed through limiting dilution byuse of a selection reagent (Zeocin).

Soluble CDH3-expressing CHO cells were selected through Western blottingby use of an anti-c-Myc monoclonal antibody (product of SANTA CRUZBIOTECHNOLOGY). Cell lines which exhibited high level of secretion intothe culture supernatant and high proliferation were selected to obtain asoluble CDH3-expressing CHO cell line (EXZ1702). The thus-selectedsoluble CDH3-expressing CHO cells (EXZ1702) were cultured for 72 hoursin three roller bottles (each culture area: 1,500 cm²) with serum-freemedium CHO-S-SFM-II (333 mL/bottle) (product of Invitrogen), and theculture supernatants were recovered. The thus-obtained culturesupernatant was subjected to affinity chromatography by means of HisTrap(registered trademark) HP column (product of GE Healthcare Bio-science)and gel filtration chromatography by means of Superdex (registeredtrademark) 200 pg column (product of GE Healthcare Bio-science), tothereby acquire soluble CDH3 protein.

Example 2 Establishment of CDH3-Expressing CHO Cell Line

For obtaining a cell line for anti-CDH3 antibody screening, a CHO cellline expressing full length CDH3 was established.

(1) Production of CDH3 Gene Expression Vector

In order to insert full-length human CDH3 DNA represented by SEQ ID NO:1 into a mammal expression vector pEF4/myc-HisB (product of Invitrogen),the full-length human CDH3 DNA was treated with two restriction enzymesKpnI (product of Takara Bio Inc.) and XbaI (product of Takara Bio Inc.)at 37° C. for one hour. The thus-obtained fragment was inserted intopEF4/myc-HisB which had been treated with the same restriction enzymesKpnI and XbaI, by use of T4 DNA ligase (product of Promega) through aroutine technique, whereby an expression vector pEF4-CDH3-myc-His wasproduced.

(2) Acquisition of Stable CDH3-Expressing Line

According to a protocol of an FuGENE (registered trademark) 6transfection reagent (product of Roche Diagnostics K.K.), 8×10⁵ CHOcells were inoculated to a 10-cm-diameter dish on the day beforetransfection, and the cells were cultured overnight. Thereafter, anexpression vector pEF4-CDH3-myc-His (8 μg) and an FuGENE6 regent (16 μL)were mixed with serum-free RPMI 1640 medium (product of SIGMA-ALDRICH)(400 μL), and the mixture was allowed to stand at room temperature for15 minutes. The resultant mixture was added to the cell culture liquidfor transfection. Two days after transfection, cloning was performedthrough limiting dilution by use of a selection reagent (Zeocin).

Clones of CDH3 full-length expressing CHO were selected through Westernblotting by use of an anti-c-Myc monoclonal antibody (product of SANTACRUZ BIOTECHNOLOGY). As a result, a CDH3 full-length expressing CHO cellline (EXZ1501) was selected as a cell line which exhibited high level ofexpression and high proliferation. The reaction between EXZ1501 and acommercial anti-CDH3 antibody (product of R&D SYSTEMS) was confirmedthrough flow cytometry. That is, CDH3 protein expression on the cellmembrane of EXZ1501 was confirmed.

Example 3 Production of Anti-CDH3 Monoclonal Antibody (1) Production ofMonoclonal Antibody by Use of Soluble CDH3 Protein as an Immunogen

Soluble CDH3 protein (50 μg) dissolved in physiological saline was mixedwith an equal amount of Titer-MAX Gold (registered trademark) (productof Titer Max), and the mixture was intraperitoneally and subcutaneouslyinjected to MRL/lpr mice (Japan SLC inc.) for initial immunization.Subsequent immunization procedures were performed by intraperitoneallyand subcutaneously injecting, to the mice, a mixture of soluble CDH3protein (25 μg) and Titer-MAX Gold prepared in the same manner. Threedays after final immunization, spleen cells were prepared from the miceunder aseptic conditions, and the cells were fused with mouse myelomacells SP2/O—Ag14 or P3-X63-Ag8.653 through a generally employedpolyethylene glycol method.

(2) Selection of Anti-CDH3 Antibody-Producing Hybridomas

Selection of anti-CDH3 antibodies were performed through flow cytometryby use of a full-length CDH3-expressing CHO cell line (EXZ1501).

Specifically, full-length CDH3-expressing CHO cells (EXZ1501) wereremoved from a culture plate by treating with 2 mM EDTA-PBS andsuspended in FACS solution to a cell concentration of 1×10⁶ cells/mL.The cell suspension was inoculated to a 96-well plate to a concentrationof 50 μL/well, and a hybridoma culture supernatant was added thereto,followed by reaction at 4° C. for 60 minutes. The plate was washed twicewith FACS solution (200 μL/well), and Alexa Fluor 488-labeled anti-mouseIgG•goat F(ab′)2 (product of Invitrogen) was added thereto, followed byreaction at 4° C. for 30 minutes. Subsequently, the plate was washedtwice with FACS solution, and flow cytometry was performed, to therebyselect hybridomas producing an antibody which binds to CDH3-expressingCHO cells. As a result, 40 clones PPMX2016 to PPAT-052-28 were obtained.Through flow cytometry, it was confirmed that all the hybridomas reactedwith CDH3-expressing CHO cells (EXZ1501) and NCI-H358 but do not reactwith CHO cells. Antibodies were purified from the hybridoma culturesupernatant by means of Protein G column and employed in the subsequentexperiments. Among the selected hybridomas, PPMX2016 (NITE BP-897),PPMX2025 (NITE BP-898), PPMX2029 (NITE BP-899), PPAT-052-02 (NITEBP-1034), PPAT-052-03 (NITE BP-1035), PPAT-052-09 (NITE BP-1036),PPAT-052-24 (NITE BP-1037), PPAT-052-25 (NITE BP-1038), PPAT-052-26(NITE BP-1039), and PPAT-052-28(NITE BP-1040) were deposited withIncorporated Administrative Agency, the National Institute of Technologyand Evaluation, Patent Microorganisms Depositary (2-5-8, Kazusakamatari,Kisarazu-shi, Chiba, Japan) on Feb. 10, 2010 and Jan. 18, 2011.

Example 4 Cloning of Antibody Genes

(1) A DNA fragment encoding the V-region of a mouse monoclonal antibodyto human CDH3 was cloned through the following procedure. CytoplasmicRNA was isolated from the mouse hybridoma cells through a methoddisclosed in a document (Gough, “Rapid and quantitative preparation ofcytoplasmic RNA from small numbers of cells,” Analytical Biochemistry,173, p. 93-95 (1988)), with the proviso that instead of the dissolutionbuffer disclosed in the document, a TNE buffer (i.e., 25 mM Tris-HCl,pH: 7.5; 1% NP-40; 150 mM NaCl; 1 mM EDTA, pH: 8.0)was employed. Morespecifically, 5×10⁶ hybridoma cells were suspended in the TNE buffer(200 μL), to thereby dissolve cell membranes, and cell nuclei wereremoved through centrifugation. To the thus-obtained cytoplasmasupernatant (about 200 μL), an extraction buffer (10 mM Tris-HCl, pH:7.5; 0.35M NaCl; 1% (w/v) SDS; 10 mM EDTA, pH: 8.0; 7M urea) (200 μL)was added. The mixture was subjected to extraction with phenol andchloroform. To the thus-obtained RNA solution, glycogen (product ofRoche, Cat No. 901393) serving as a carrier was added. Then, ethanol wasadded to precipitate the product. The RNA precipitate was dissolved insterilized distilled water (10 to 50 μL) to a cytoplasmic RNAconcentration of 0.5 to 2 μg/μL.(2) Production of cDNA Library from RNA Prepared from Hybridomas

For synthesizing a single-strand cDNA, there was prepared a reactionmixture (20 μL) containing the above-prepared cytoplasmic RNA (0.5 to 3μg), 50 mM Tris-HCl (pH: 8.3, room temperature), 75 mM KCl, 3 mM MgCl₂,and 10 mM DTT), a random primer (100 ng), 0.5 mM dNTP, and SuperscriptII (reverse transcriptase, product of Invitrogen) (200 units). Themixture was incubated at 42° C. for 50 minutes. The thus-synthesizedcDNA library was employed as a template of polymerase chain reaction(PCR) without performing further treatment.

(3) Amplification of a Gene Encoding a Variable Region of Anti-CDH3Antibody Through PCR

All the primers employed in the experiments were synthesized by HokkaidoSystem Science Co., Ltd.

a. Primers for Use in PCR Amplification of a Gene Encoding Mouse L-ChainV-Region

The following two primer sets were employed: (i) a DNA primer having, atthe 5′ end, a homology to the FR1 part and 4-set primers having, at the3′ end, a homology to a J-chain gene in the mouse L-chain, and (ii)7-set primers having, at the 5′ end, a homology to the L-chain signalpart and an antisense primer having, at the 3′ end, a homology to the KCpart (KVL antisense primer). Polymerase chain reaction was performed byuse of the two primer sets, whereby a mouse immunoglobulin L-chainvariable region DNA fragment was obtained from the cDNA. The primersequences are as follows.

(i) 4-Set Sense Primers for Mouse L-Chain Variable Region Cloning

According to “Phage Display—A Laboratory Manual-, Barbas Burton ScottSilverman,” PROTOCOL 9.5, 17 sense primers and 3 reverse primers weresynthesized by Hokkaido System Science Co., Ltd.

VK Sense (FR1 Part)

A mixture of the following 17 primers was employed as a VK sense primer.

(degeneracy 2): SEQ ID NO: 5 5′-GAY ATC CAG CTG ACT CAG CC-3′(degeneracy 4): SEQ ID NO: 6 5′-GAY ATT GTT CTC WCC CAG TC-3′(degeneracy 8): SEQ ID NO: 7 5′-GAY ATT GTG MTM ACT CAG TC-3′(degeneracy 8): SEQ ID NO: 8 5′ GAY ATT GTG YTR ACA CAG TC-3′(degeneracy 8): SEQ ID NO: 9 5′ GAY ATT GTR ATG ACM CAG TC-3′(degeneracy 16): SEQ ID NO: 10 5′ GAY ATT MAG ATR AMC CAG TC-3′(degeneracy 12): SEQ ID NO: 11 5′ GAY ATT CAG ATG AYD CAG TC-3′(degeneracy 4): SEQ ID NO: 12 5′ GAY ATY CAG ATG ACA CAG AC-3′(degeneracy 4): SEQ ID NO: 13 5′ GAY ATT GTT CTC AWC CAG TC-3′(degeneracy 8): SEQ ID NO: 14 5′ GAY ATT GWG CTS ACC CAA TC-3′(degeneracy 16):  SEQ ID NO: 15 5′ GAY ATT STR ATG ACC CAR TC-3′(degeneracy 16): SEQ ID NO: 16 5′ GAY RTT KTG ATG ACC CAR AC-3′(degeneracy 12): SEQ ID NO: 17 5′ GAY ATT GTG ATG ACB CAG KC-3′(degeneracy 4): SEQ ID NO: 18 5′ GAY ATT GTG ATA ACY CAG GA-3′(degeneracy 4): SEQ ID NO: 19 5′ GAY ATT GTG ATG ACC CAG WT-3′(degeneracy 2): SEQ ID NO: 20 5′ GAY ATT GTG ATG ACA CAA CC-3′(degeneracy 2): SEQ ID NO: 21 5′ GAY ATT TTG CTG ACT CAG TC-3′

J Antisense (4-Set Primers) J1/J2 Antisense Primer (1)

(degeneracy 8): SEQ ID NO: 22 5′-GGS ACC AAR CTG GAA ATM AAA-3′

J4 Antisense Primer (2)

SEQ ID NO: 23 5′-GGG ACA AAG TTG GAA ATA AAA-3′:

J5 Antisense Primer (3)

SEQ ID NO: 24 5′-GGG ACC AAG CTG GAG CTG AAA-3′: 

J1/J2, J4, J5 Antisense Primer Mixture (4) (ii) 7-Set Primers for MouseL-Chain Variable Region Cloning VK Sense (Signal Peptide Part)

The primers were obtained through nucleotide sequence modification of amouse Ig-primer set (Novagen; Merck, Cat. No. 69831-3) such thatrestriction enzyme sites were removed. A-set sense primer

SEQ ID NO: 25 5′-ATGRAGWCACAKWCYCAGGTCTTT-3′: 

B-Set Sense Primer

SEQ ID NO: 26 5′-ATGGAGACAGACACACTCCTGCTAT-3′:

C-Set Sense Primer

SEQ ID NO: 27 5′-ATGGAGWCAGACACACTSCTGYTATGGGT-3′:

D-Set Sense Primer (Mixture of the Following 2 Primers)

SEQ ID NO: 28 5′-ATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT-3′: SEQ ID NO: 295′-ATGGGCWTCAAGATGRAGTCACAKWYYCWGG-3′:

E-Set Sense Primer (Mixture of the Following 3 Primers)

SEQ ID NO: 30 5′-ATGAGTGTGCYCACTCAGGTCCTGGSGTT-3′: SEQ ID NO: 315′-ATGTGGGGAYCGKTTTYAMMCTTTTCAATTG-3′: SEQ ID NO: 325′-ATGGAAGCCCCAGCTCAGCTTCTCTTCC-3′:

F-Set Sense Primer (Mixture of the Following 4 Primers)

SEQ ID NO: 33 5′-ATGAGIMMKTCIMTTCAITTCYTGGG-3′: SEQ ID NO: 345′-ATGAKGTHCYCIGCTCAGYTYCTIRG-3′: SEQ ID NO: 355′-ATGGTRTCCWCASCTCAGTTCCTTG-3′: SEQ ID NO: 365′-ATGTATATATGTTTGTTGTCTATTTCT-3′: 

G-Set Sense Primer (Mixture of the Following 4 Primers)

SEQ ID NO: 37 5′-ATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3′: SEQ ID NO: 385′-ATGGATTTWCARGTGCAGATTWTCAGCTT-3′: SEQ ID NO: 395′-ATGGTYCTYATVTCCTTGCTGTTCTGG-3′: SEQ ID NO: 405′-ATGGTYCTYATVTTRCTGCTGCTATGG-3′:

KVL Antisense Primer

SEQ ID NO: 41 ACTGGATGGTGGGAAGATGGA: 

B. Primers for Use in PCR Amplification of a Gene Encoding Mouse H-ChainV-Region

The following two primer sets were employed 4-set primers having, at the5′ end, a homology to the mouse H-chain signal part and a primer having,at the 3′ end, a homology to the KC part; and 1-set primers having, atthe 5′ end, a homology to the FR1 part and 2-set primers having, at the3′ end, a homology to the mouse H-chain constant region (IGHC).Polymerase chain reaction was performed by use of the two primer sets,whereby a mouse immunoglobulin H-chain variable region DNA fragment wasisolated from the cDNA. The primer sequences are as follows.

(i) Primers for Mouse H-Chain Variable Region Cloning VH Sense (SignalPart: 4-Set Primers)

These primers were synthesized according to Current Protocols inImmunology (John Wiley and Sons, Inc.), Unit 2.12 Cloning, Expression,and Modification of Antibody V Regions (Table 2.12.2).

(degeneracy: 32): SEQ ID NO: 42 5′-ATG GRA TGS AGC TGK GTM ATS CTC TT-3′(degeneracy: 8): SEQ ID NO: 43 5′-ATG RAC TTC GGG YTG AGC TKG GTT TT-3′SEQ ID NO: 44 5′-ATG GCT GTC TTG GGG CTG CTC TTC T-3′: (degeneracy: 32): SEQ ID NO: 45 5′-ATG GRC AGR CTT ACW TYY-3′

(ii) Primers for Mouse H-Chain Variable Region Cloning VH Sense (FR1Part)

These primers were designed by nucleotide sequence modification of senseprimers disclosed in a document (Tan et al, “Superhumanized” Antibodies:Reduction of Immunoogenic Potential by Complementarity-DeterminingRegion Grafting with Human Germline Sequences: Application to anAnti-CD281, Journal of Immunology 169 (2002), p. 1119-1125).

(degeneracy: 256): SEQ ID NO: 46 5′-SAG GTS MAR CTK SAG SAG TCW GG-3′

VH Antisense (Antisense Primer Common to 3 and 4)

The primer was designed through degeneration of the nucleotide sequenceso that the primer can be annealed with all the isoforms of mouse IgG.

(degeneracy: 6): SEQ ID NO: 47 5′-CAS CCC CAT CDG TCT ATC C-3′

Example 5 Production of Chimera Anti-CDH3 Immunoglobulin ExpressionVector Production of Expression Plasmid

Through PCR employing DNA Engine (Peltier Thermal Cycler, MJ Research,Bio-Rad), each variable region of the L-chain and the H-chain of ananti-CDH3 mouse monoclonal antibody was amplified by use of the primersdescribed in Example 4. Each of the thus-amplified DNA fragments wasincorporated into a sub-cloning vector pGEM (product of Promega). Thenucleotide sequence of the DNA fragment was determined by use of auniversal primer which binds to T7 an 6. SP6 promoter of the vector. Thethus-obtained nucleotide sequences of the L-chain and H-chain variableregions of the anti-CDH3 antibody were searched by IMGT/V-QUEST Searchpage (http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=mouseIg),whereby completion of cloning of the antibody genes was confirmed.

Next, a gene encoding the human Cκ region was linked to the cloned geneencoding the V region of the L-chain of the anti-CDH3 antibody, and agene encoding the human Cκ1 region was linked to the gene encoding the Vregion of the H-chain. The thus-designed L-chain and H-chain chimericantibody genes were synthesized in full length by GenScript. At thetime, frequency of codon usage was optimized so as to obtain efficientgene expression in producing cells (according to a method disclosed inKim et al., Codon optimization for high-level expression of humanerythropoietin (EPO) in mammalian cells, Gene, 199, 1997, p. 293-301).Specifically, in the case of L-chain, for the purpose of effectivetranslation, an essential DNA sequence (Kozak, M., J., At least sixnucleotides preceding the AUG initiator codon enhance translation inmammalian cells. J. Mol. Biol. 196, p. 947-950, 1987), signal peptide ofmouse IGKV, the V region of the L-chain of the anti-CDH3 antibody, andthe human Cκ region were juxtaposed in this order, and restrictionenzyme sites were added to both ends (NheI on the 5′ side and EcoRI onthe 3′ side). The chimera H-chain was prepared in the same manner. Eachof the synthesized genes was cut with NheI and EcoRI, and the cutfragment was incorporated into an expression vector pCAGGS between theNheI site and the EcoRI site, to thereby produce an anti-CDH3 chimericantibody L-chain expression vector pCAGGS-IGK and H-chain expressionvector pCAGGS-IGH.

Example 6 Production of Chimera Anti-CDH3 Immunoglobulin StableExpression Vector

For realizing high-level expression of a genetically modified antibodygene in CHO cells, there was produced an expression vector into which adihydrofolate reductase (dhfr) gene linked to a CMV promoter sequenceand having poly A signal had been incorporated.

For producing a chimeric antibody-stably expressing/producing cell line,there was produced a pCAGGS expression vector into which a dhfr gene hadbeen incorporated. Specifically, into pCAGGS-IGH and pCAGGS-IGK, whichare transient expression vectors, a dhfr gene having a CMV promoter andpoly A signal was incorporated. Each of a mouse dhfr gene having a CMVpromoter and a Kozak sequence and SV40 poly A signal was amplifiedthrough PCR. These genes in mixture form were linked together throughPCR, and an HindIII site was added to both ends of the linked product,to thereby acquire a gene fragment of HindIII-CMVpromoter-Kozak-dhfr-poly A-HindIII. The fragment was inserted intopCAGGS-IGH or pCAGGS-IGK at the HindIII sites, to thereby obtainpCAGGS-IGH-CMVp-dhfr-A and pCAGGS-IGK-CMVp-dhfr-A. These expressionvectors enable chimeric antibody expression with a CAG promoter, andexpression of a dhfr gene with a CMV promoter, whereby a chimericantibody can be effectively produced through gene amplification.

Example 7 Establishing a Chimera Anti-CDH3-Producing CHO Cell Line

CHO dhfr cells (G. Urlaub et al., Isolation of Chinese hamster cellmutants deficient in dihydrofolate reductase activity, Proc. Natl. Acad.Sci. USA 77, p. 4216-4220, 1980) were simultaneously transformed by useof two plasmids (linear plasmids obtained by cutting circular plasmidswith PvuI in an ampicillin-resistant gene); i.e., apCAGGS-IGK-CMV-dhfr-A vector for chimera anti-CDH3 L chain expressionand a pCAGGS-IGH-CMV-dhfr-A vector for chimera anti-CDH3 H chainexpression. Electroporation was performed by means of Amaxa (product ofLonza). DNA fragment (2 μg/sample; in the case of L-chain plasmid orH-chain plasmid) was added to a 0.1 mL Amaxa electroporation CHO buffercontaining 3×10³ cells and a pulse was applied.

The cells which had undergone electroporation were added to an Iscove'sModified Dulbecco medium (IMDM: free of HT) containing 10% dialyzed FBSthat is free of HT (H: hypoxanthine, T: thymidine). Three days aftertransfection, the medium was changed to an IMDM medium free of 10%dialyzed FBS, 2 mM L-glutamine, and HT, and neo+ transformed cells wereselected by use of 1 mg/mL G418, to thereby acquire clones of a chimericantibody-producing positive cell line. Subsequently, gene amplificationwas performed by use of the clones selected by using G418. 2-Roundamplification was performed in 250 nM and 1,000 nM methotrexate (MTX),and cell lines which can produce a chimera CDH3 antibody (about 50 to100 mg/L-culture supernatant) were established. The thus establishedchimera anti-CDH3 antibody-stably expressing CHO cell lines weredeposited with Incorporated Administrative Agency, the NationalInstitute of Technology and Evaluation, Patent MicroorganismsDepositary.

TABLE 1 Cell line Accession No. PPAT-052-02c NITE BP-1041 PPAT-052-03cNITE BP-1042 PPAT-052-09c NITE BP-1043 PPAT-052-21c NITE BP-1044PPAT-052-24c NITE BP-1045 PPAT-052-25c NITE BP-1046 PPAT-052-26c NITEBP-1047 PPAT-052-27c NITE BP-1048 PPAT-052-28c NITE BP-1049 PPAT-052-29cNITE BP-1050

Example 8 Acquisition of Purified Antibodies

The antibodies were purified from the culture supernatant by use ofprotein A.

Example 9 Confirmation of Affinity

Through a competitive method, the affinity of the mouse anti-CDH3antibody was compared with that of the chimera anti-CDH3 antibody. Inthe competitive method, the affinity of the anti-CDH3 antibody wasdetermined through flow cytometry (BD, FACS Calibur) by use of cancercells NCI-H358, which are known to be CDH3 high expression cells.

Specifically, an antibody serially diluted sample (400 μg/mL to 24ng/mL) (50 μL) and an Alexa488-labeled antibody (4 μg/mL) (50 μL) wereadded to and mixed on a 96-well plate. NCI-H358 cells were removed froma culture plate through treatment with 2 mM EDTA-PBS, and the cells weresuspended in an FACS solution (1% BSA PBS) to a concentration of1.5×10⁶/mL. An aliquot (100 μL) of the suspension was added to the wellscontaining the antibody mixture. After addition, reaction was performedat room temperature for 60 minutes, and the plate was washed twice withan FACS solution (200 μL/well). Subsequently, the fluorescence intensity(GEO mean) of each well was determined through flow cytometry.

The percent of binding inhibition of the Alexa488-labeled antibody wascalculated from a GEO mean value, as compared with that obtained by thereaction only with the Alexa488-labeled antibody (1 pg/mL). The antibodyconcentration showing 50% inhibition was calculated, and the data werecompared.

FIG. 1 shows the affinity evaluation of PPMX2016 (mouse antibody) andPPAT-052-27c (chimeric antibody thereof). Virtually no difference inaffinity was observed between the two antibodies.

Example 10 Production of Labeled Antibodies (1) Bonding DOTA to Antibody

An antibody was dissolved in a buffer (50 mM Bicine-NaOH, 150 mM NaCl,pH: 8.5) to an antibody concentration of 10 mg/mL. Separately,isothiocyanobenzyl DOTA (B-205, product of Macrocyclics) was dissolvedin DMSO to a concentration of 10 mg/mL. The two solutions were mixedtogether so as to adjust the ratio by mole of antibody to DOTA 1:1(adding ratio 1:1), 1:3 (adding ratio 1:3), or 1:10 (adding ratio 1:10),and the mixture was stirred and allowed to stand at 25° C. for 17 hours.After termination of reaction, the reaction mixture was purified bymeans of a desalting column (PD-10, product of GE Healthcare,17-0435-01) with PBS. The following antibodies were used: PPMX2016,PPMX2025, PPMX2029, PPAT-052-27c, and PPAT-052-28c.

(2) Determination of Percent of Chelate Incorporation

The percent of chelate incorporated into antibody was determined throughchelatometric titration. The modification antibody protein concentrationwas determined through a customary method in advance, and the number ofmoles of modification antibody was calculated from the molecular weightof IgG. To 1-mg/mL standard copper solution (100 whose concentration hadbeen determined through atomic absorption spectrometry, an arsenazo IIIreagent (0.776 mg) and metal-free 5M ammonium acetate (product of SigmaAldrich) solution (3 mL) were added, and ultrapure water was added tothe solution to adjust the final volume to 10 mL. The resultant solutionwas stored at room temperature in the dark to prepare the arsenazo IIIsolution. DOTA was dissolved in ultrapure water, to thereby prepare aDOTA standard solution. A modification antibody was dissolved inultrapure water, to thereby prepare a modification antibody solution.The DOTA standard solution or the modification antibody solution (10 μL)was admixed with the arsenazo III solution (190 μL), and the mixture wasincubated at 37° C. for 30 minutes. Subsequently, the absorbance of themixture was measured at a wavelength of 630 nm. A standard curve wasdrawn from the absorbance measurements of DOTA standard solutions. Bythe standard curve, the number of DOTA molecule(s) bound to themodification antibody was calculated (average number of DOTAmodification).

Table 2 shows the results (DOTA-adding ratio and actual average numberof modifying DOTA). As is clear from Table 2, the number of DOTAmolecules bound to the antibody was found to be determined by the addingratio of DOTA.

TABLE 2 Av. no. of Antibody to DOTA-adding ratio modifying DOTA PPMX2025(adding ratio 1:1) 0.9 PPMX2025 (adding ratio 1:3) 2.0 PPMX2025 (addingratio 1:10) 5.3 PPMX2016 (adding ratio 1:1) 0.7 PPMX2016 (adding ratio1:3) 2.1 PPMX2016 (adding ratio 1:10) 5.2 PPAT-052-27c (addding ratio1:3) 1.8 (Note) PPAT-052-27c was tested only at an adding ratio of 1:3.

(3) Preparation of ⁶⁷Ga-, or ⁹⁰Y-Labeled Antibodies

(i) Labeling with ⁶⁷Ga or ¹¹¹In

Each of the purified PPMX2016, PPMX2025, PPMX2029, and PPAT-052-27cantibodies and PPAT-052-28c antibody was dissolved in a buffer (0.25Mammonium acetate-HCl, pH: 5.5) to a concentration of 6 mg/mL. A ⁶⁷GaCl₃solution (product of Fuji Film RI Pharma) or a ¹¹¹InCl₃ solution(product of MDS Nordion Inc.) was added to the antibody solution, andthe mixture was incubated at 45° C. for one hour.

(ii) Labeling with ⁹⁰Y

Each of the purified PPMX2029 and PPAT-052-27c antibodies was dissolvedin a buffer (0.25M ammonium acetate-HCl, pH: 5.5) to a concentration of6 mg/mL. A ⁹⁰YCl₃ solution (product of Nuclitec) was added to theantibody solution, and the mixture was incubated at 45° C. for one hour.

(iii) Determination of Percent of Labeling

An aliquot of the labeling reaction mixture was sampled and subjected tothin-layer chromatography (61885, product of PALL), to thereby determinethe percent of labeling. Physiological saline was employed as an eluent,and the radioactivity was measured at the top and bottom ends of a stripby means of a γ-counter. The percent of labeling was calculated by thefollowing equation.

Percent of labeling=(bottom end count/(top end count+bottom endcount))×100(%)

When the percent of labeling reached 90% or higher, the labeled antibodywas used in the subsequent experiment. The labeled antibodies werepurified a desalting column (PD-10, product of GE Healthcare,17-0435-01) with PBS.

Example 11 Investigation of Relationship Between Percent ofChelate-Incorporation and Bio-Distribution (i.e., Distribution in theBody)

PPMX2016, PPMX2025, and PPMX2029 were investigated in terms ofbio-distribution by virtue of difference of percent of chelateincorporation values (DOTA-adding ratio 1:1, 1:3, and 1:10).

Firstly, NCI-H358 was cultured in a 10% FBS-containing RPMI1640 medium,and the cultured cells were subcutaneously transplanted to the rightventral region of each of the nude mice (female, 7-week old, CLEA JapanInc.) at a cell concentration of 1×10⁷ cells/mouse. The mice were breduntil the average tumor volume reached 100 to 150 mm³.

Then, to the NCI-H358-transplanted mice, ⁶⁷Ga-DOTA-PPMX2016 antibody(adding ratio 1:3 and 1:10), ⁶⁷Ga-DOTA-PPMX2025 antibody (adding ratio1:3 and 1:10), or ⁶⁷(Ga-DOTA-PPMX2029 antibody(adding ratio 1:3 and1:10) was administered at a dose of 370 kBq/mouse.

Ninety-six hours after administration, the mice were sacrificed toanatomy, and tissues and the tumor were removed. The weight of eachtissue and the tumor weight were measured, the radioactivity wasmeasured by means of a γ-counter and % ID/g was calculated by thefollowing equation.

% ID/g=(accumulated RI amount/total administered RIamount×100(%))/weight (g)  [E2]

FIGS. 2 to 7 show the results. All the tested antibodies exhibitedenhanced accumulation in the tumor, at a DOTA-adding ratio of 1:3, ascompared with the case at a ratio of 1:10. Such enhanced accumulationresults in enhancement of therapeutic effect. In addition, adverse sideeffects, which would otherwise be caused by retention of a radioactivesubstance in non-targeted organs, can be avoided.

FIG. 8 shows the result of administration of ⁶⁷Ga-DOTA-PPMX2025 antibody(use ratio 1:1, 1:3, and 1:10) to non-cancer-bearing nude mice (female,7-week old, CLEA Japan Inc.) at 370 kBq/mouse. The antibody wasaccumulated highly in the liver when the adding ratio was 1:10, whereasaccumulation in the liver was low when the ratio was 1:3 or 1:1, whichindicates that adverse side effects such as radioactive damage areprevented on the liver.

Example 12 Investigation of Behavior of Anti-Caldina Chimeric Antibodyin the Body

The behaviors of chimera antibodies PPAT-052-27c and PPAT-052-28c in thebody were investigated.

Firstly, NCI-H1373 was cultured in a 10% FBS-containing RPMI1640 medium,and the cultured cells were subcutaneously transplanted into to theright ventral region of each of the nude mice (female, 9-week old, CLEAJapan Inc.) at a cell concentration of 4×10⁶ cells/mouse. The mice werebred until the average tumor volume reached 100 to 150 mm³.

Then, to the NCI-H1373-transplaned mice, ¹¹¹In-DOTA-PPAT-052-27c (addingratio 1:3) or ¹¹¹In-DOTA-PPAT-052-28c (adding ratio 1:3) at a dose of370 kBq/mouse.

Forty-eight or ninety-six hours after administration, the mice weresacrificed to anatomy, and tissues and the tumor were removed. Theweight of each tissue and the tumor weight were measured, theradioactivity was measured by means of a γ-counter and % ID/g wascalculated.

FIGS. 9 to 11 show the results. PPAT-052-27c exhibited a percentaccumulation in the tumor as high as 46% ID/g 48 hours afteradministration. PPAT-052-28c exhibited a percent accumulation in thetumor as high as 41% ID/g 48 hours after administration and 52% ID/g 96hours after administration.

Example 13 Xenograft Test

NCI-H358 was cultured in a 10% FBS-containing RPMI1640 medium, and thecultured cells were subcutaneously transplanted into to the rightventral region of each of the nude mice (female, 7-week old, CLEA JapanInc.) at a cell concentration of 1×10⁷ cells/mouse.

The NCI-H358-transplaned mice were divided into six groups (n=8).⁹⁰Y-DOTA-PPMX2029 antibody (adding ratio 1:3) was administered at a doseof 7.4 MBq/mouse, 5.6 MBq/mouse, 3.7 MBq/mouse, and 1.9 MBq/mouse. Ascontrol groups, unlabeled PPMX2029 was administered at a dose of 80μg/mouse, and physiological saline was administered at a dose of 100μL/mouse. In all the groups, administration was performed when theaverage tumor volume reached 100 to 150 mm³.

After administration, the body weight and the tumor volume were measuredtwice a week (every 3 or 4 days). This observation wad continued to day51 after administration.

FIG. 12 shows the test results. ⁹⁰Y-DOTA-PPMX2029 antibody (adding ratio1:3) exhibited anti-tumor effect proportional to the radioactivity.

Separately, NCI-H1373 was cultured in a 10% FBS-containing RPMI1640medium, and the cultured cells were subcutaneously transplanted into tothe right ventral region of each of the nude mice (female, 7-week old,CLEA Japan Inc.) at a cell concentration of 5×10⁶ cells/mouse.

The NCI-H1373-transplaned mice were divided into four groups (n=8).⁹⁰Y-DOTA-PPAT-052-27c antibody (adding ratio 1:3) was administered at adose of 5.6 MBq/mouse and 3.7 MBq/mouse. As control groups, unlabeledPPMX2029 was administered at a dose of 60 μg/mouse, and physiologicalsaline was administered at a dose of 100 μL/mouse. In all the groups,administration was performed when the average tumor volume reached 100to 150 mm³.

After administration, the body weight and the tumor volume were measuredtwice a week (every 3 or 4 days). This observation wad continued to day26 after administration.

FIG. 13 shows the test results. ⁹⁰Y-DOTA-PPAT-052-27c (adding ratio 1:3)exhibited anti-tumor effect proportional to the radioactivity.

Example 14 Immunohistochemical Staining

CDH3 protein expression in a clinical cancer specimen was confirmed byimmunohistochemical staining of a cancer specimen tissue array.

As cancer specimen tissue arrays, employed were tissues of pancreaticcancer (adenocarcinoma), lung cancer (adenocarcinoma), lung cancer(squamous cell carcinoma), and colorectal cancer (adenocarcinoma), whichare the products of Shanghai Outdo Biotech Co., Ltd.

Each tissue array slide was dewaxed and activated with 10 mM Tris 1 mMEDTA (pH: 9.0) at 95° C. for 40 minutes. Endogenous peroxidase in thearray slide was inactivated with a blocking agent, which is included inthe ENVISION+Kit (product of Dako). Subsequently, the tissue array slidewas reacted with 5 μg/mL anti-CDH3 antibody 610227 (product of BDBIOSCIENCE) or with 5 μg/mL anti-HBs antibody Hyb-3423 (negativecontrol) at 4° C. overnight. The antibody solution was washed out, andthe tissue array slide was further reacted with a polymer secondaryantibody reagent which is included in the ENVISION+Kit at roomtemperature for 30 minutes. The slide was then color-developed by acoloring reagent which is included in the ENVISION+Kit, and nuclearstaining was performed by use of a hematoxylin/eosin solution.

FIG. 14 shows the results. Cancer cells were stained by anti-CDH3antibody, but normal cells were not stained.

1: A radioactive metal-labeled anti-cadherin antibody, which is obtainedby binding a radioactive metallic element to an anti-cadherin antibodyvia a metal-chelating reagent, wherein a mole ratio of theanti-p-cadherin antibody to the metal-chelating reagent is from 1:0.1 to1:4.5. 2: The radioactive metal-labeled anti-p-cadherin antibody ofclaim 1, wherein the mole ratio of the anti-p-cadherin antibody to themetal-chelating reagent is from 1:0.5 to 1:3. 3: The radioactivemetal-labeled anti-p-cadherin antibody of claim 1, wherein theanti-p-cadherin antibody is bonded to a region 1 to 655 of SEQ ID NO:2.4: The radioactive metal-labeled anti-p-cadherin antibody of claim 1,wherein the metal-chelating reagent is selected from the groupconsisting of isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA andcyclohexylisothiocyanobenzyl DTPA. 5: The radioactive metal-labeledanti-p-cadherin antibody of claim 1, wherein the anti-p-cadherinantibody is a monoclonal antibody, a recombinant antibody, a chimericantibody thereof, a humanized antibody, or a fragment thereof. 6: Theradioactive metal-labeled anti-p-cadherin antibody of claim 1, whereinthe radioactive metallic element is cytotoxic radioactive metal suitablefor treating a p-cadherin expressing cancer. 7: The radioactivemetal-labeled anti-p-cadherin antibody of claim 6, wherein the cytotoxicradioactive metal is selected from the group consisting of yttrium-90(⁹⁰Y), rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re), copper-67 (⁶⁷Cu),iron-59 (⁵⁹Fe), strontium-89 (⁸⁹Sr), gold-198 (¹⁹⁸Au), dysprosium-165(¹⁶⁵Dy), ruthenium-103 (¹⁰³Ru), holmium-166 (¹⁶⁶Ho), samarium-153(¹⁵³Sm), and lutetium-177 (¹⁷⁷Lu). 8: The radioactive metal-labeledanti-p-cadherin antibody of claim 6, wherein the cytotoxic radioactivemetal is yttrium-90 (⁹⁰Y). 9: The radioactive metal-labeledanti-p-cadherin antibody of claim 1, wherein the radioactive metallicelement is non-cytotoxic radioactive metal suitable for diagnosing ap-cadherin expressing cancer. 10: The radioactive metal-labeledanti-p-cadherin antibody of claim 9, wherein the non-cytotoxicradioactive metal is selected from the group consisting oftechnetium-99m (^(99m)-Tc), indium-111 (¹¹¹In), indium-113m (^(113m)In),gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga), thallium-201 (²⁰¹Tl), cobalt-57(⁵⁷Co), strontium-85 (⁸⁵Sr), and copper-64 (⁶⁴Cu). 11: The radioactivemetal-labeled anti-p-cadherin antibody of claim 9, the non-cytotoxicradioactive metal is indium-111 (¹¹¹In) or copper-64 (⁶⁴Cu). 12: Ap-cadherin expressing cancer therapeutic agent comprising as an activeingredient the radioactive metal-labeled anti-p-cadherin antibody ofclaim
 6. 13: A p-cadherin expressing cancer diagnosing agent comprisingas an active ingredient the radioactive metal-labeled anti-p-cadherinantibody of claim
 9. 14: A metal-chelating reagent-bindinganti-p-cadherin antibody, obtained by a process comprising binding ametal chelating reagent to an anti-p-cadherin antibody, wherein a moleratio of the anti-p-cadherin antibody to the metal-chelating reagent isfrom 1:0.1 to 1:4.5. 15: The metal chelating reagent-bindinganti-p-cadherin antibody of claim 14, wherein the mole ratio of theanti-p-cadherin antibody to the metal-chelating reagent is from 1:0.5 to1:3. 16: The metal chelating reagent-binding anti-p-cadherin antibody ofclaim 14, wherein the anti-p-cadherin antibody is bonded to a region 1to 655 of SEQ ID NO:2 17: The metal chelating reagent-bindinganti-p-cadherin antibody of claim 14, wherein the metal-chelatingreagent is selected from the group consisting of isothiocyanobenzylDOTA, methylisothiocyanobenzyl DTPA and cyclohexylisothiocyanobenzylDTPA. 18: The metal chelating reagent-binding anti-p-cadherin antibodyof claim 14, wherein the anti-p-cadherin antibody is a monoclonalantibody, a recombinant antibody, a chimeric antibody, a humanizedantibody, or a fragment thereof. 19: A kit suitable for preparing aradioactive metal-labeled anti-p-cadherin antibody, comprising a metalchelating reagent-binding anti-p-cadherin antibody of claim
 14. 20: Thekit of claim 19, wherein the anti-p-cadherin antibody is a monoclonalantibody produced by an antibody-producing cell of an accession numberof NITE BP-897, NITE BP-898, NITE BP-899, NITE BP-1040, NITE BP-1044,NITE BP-1048, NITE BP-1049 or NITE BP-1050, or a recombinant antibodythereof or a chimeric antibody thereof or a humanized antibody thereof,or a fragment of any of these antibodies. 21: A method for producing ametal-chelating reagent-binding anti-p-cadherin antibody; comprisingadding an anti-p-cadherin antibody and a metal-chelating reagent at aratio of 1:0.1 to 1:less than 5 to react. 22: The method of claim 21,wherein the anti-p-cadherin antibody and the metal chelating reagent areadded at a ratio of 1:1 to 1:less than 3 to react. 23: A method fortreating a p-cadherin expressing cancer, comprising: administering aneffective amount of the radioactive metal-labeled anti-p-cadherinantibody of claim 6 to a subject in need thereof. 24: A method fordiagnosing a p-cadherin expressing cancer, comprising: administering aneffective amount of the radioactive metal-labeled anti-p-cadherinantibody of claim 9 to a subject in need thereof; and diagnosing thep-cadherin expressing cancer.